Cloning and Functional Analysis of CsROP5 and CsROP10 Genes Involved in Cucumber Resistance to Corynespora cassiicola

Simple Summary The CsROP gene plays a critical role in the regulation of defense responses. We cloned the cucumber genes CsROP5 and CsROP10 and identified their structure domains containing two Rho-related guanosine triphosphatases. The CsROP5 and CsROP10 genes negatively contributed to defense resistance to Corynespora cassiicola by regulating the ROS signaling pathway, the ABA signaling pathway, and the PR gene. Abstract The cloning of resistance-related genes CsROP5/CsROP10 and the analysis of their mechanism of action provide a theoretical basis for the development of molecular breeding of disease-resistant cucumbers. The structure domains of two Rho-related guanosine triphosphatases from plant (ROP) genes were systematically analyzed using the bioinformatics method in cucumber plants, and the genes CsROP5 (Cucsa.322750) and CsROP10 (Cucsa.197080) were cloned. The functions of the two genes were analyzed using reverse-transcription quantitative PCR (RT-qPCR), virus-induced gene silencing (VIGS), transient overexpression, cucumber genetic transformation, and histochemical staining technology. The conserved elements of the CsROP5/CsROP10 proteins include five sequence motifs (G1-G5), a recognition site for serine/threonine kinases, and a hypervariable region (HVR). The knockdown of CsROP10 through VIGS affected the transcript levels of ABA-signaling-pathway-related genes (CsPYL, CsPP2Cs, CsSnRK2s, and CsABI5), ROS-signaling-pathway-related genes (CsRBOHD and CsRBOHF), and defense-related genes (CsPR2 and CsPR3), thereby improving cucumber resistance to Corynespora cassiicola. Meanwhile, inhibiting the expression of CsROP5 regulated the expression levels of ROS-signaling-pathway-related genes (CsRBOHD and CsRBOHF) and defense-related genes (CsPR2 and CsPR3), thereby enhancing the resistance of cucumber to C. cassiicola. Overall, CsROP5 and CsROP10 may participate in cucumber resistance to C. cassiicola through the ROS and ABA signaling pathways.


Introduction
Corynespora cassiicola is a pathogenic fungus with a wide host range.It causes cucumber target leaf spot (TLS), which is a significant leaf disease in cucumber production [1].Cassiicolin is the main pathogenic factor of the fungus, which is a small, secreted glycoprotein consisting of six different subtypes: Cas1, Cas2, Cas3, Cas4, Cas5, and Cas6 [2][3][4][5][6].Currently, there is an increasing amount of research on the molecular mechanisms of the interaction between cucumber and C. cassiicola.Cucumbers with C. cassiicola resistance include the caffeoyl shikimate esterase gene (CsCSE5) [7].CsSnRK1 overexpression improves cucumber resistance to C. cassiicola, and CsSnRK1 silencing reduces the resistance of cucumber to C. cassiicola [8].Two cucumber genes, CsMLO1 and CsMLO2, negatively Biology 2024, 13, 308 2 of 16 regulate its resistance to C. cassiicola [9].Therefore, we speculate that complex networks form during pathogen infection in plants, and different signaling pathways are related to the transcription of some genes in their mediated disease-resistance responses.Research has confirmed that plant Rho-type (ROP) small G proteins are involved in various signal transduction processes, including regulating pollen tube growth and root hair growth and responding to biotic and abiotic stresses [10][11][12][13][14][15].However, the molecular mechanism of CsROP in regulating cucumber resistance to cucumber TLS remains unclear.
Small G protein is a GTP-binding protein that contains an active guanosine triphosphatase (GTPase).According to their functional differences, small G proteins can be categorized into five families: Ras, Rho, Rab, Ran, and Arf.ROP (Rho-related GTPase from plants) belongs to the small G protein Rho subfamily and is a very conserved signaling molecule [16,17].Yang and other scholars proposed a nomenclature system in 2002 to unify the ROP GTPase in Arabidopsis as ROPs [18].The highly conserved ROP family members have four GTP/GDP-binding domains and one domain that binds to downstream effector proteins [19].ROP plays a molecular switch role in plant signal transduction, similar to other small G proteins.The GDP-bound inactive form is mediated by guanine nucleotide exchange factor (GEF) to transform into the GTP-bound active form [20]. Small G proteins in the GTP-binding state have only weak GTPase hydrolysis activity, which requires a GTPase-activated protein (GAP), which can efficiently inactivate ROP in the GTP state and convert it into GDP, releasing inorganic phosphorus.Guanine nucleotide dissociation inhibitor (GDI) in the cytoplasm can bind to the C-terminal lipid modification of ROPs, thereby affecting their membrane localization [20].The activation of ROP is mediated by upstream signals, and ROP also activates downstream targets.The ROP molecular switch can activate numerous downstream cellular activities, including the dynamic assembly of the microfilament cytoskeleton, calcium ion fluctuations, ROS production, and vesicle transport [21][22][23][24].Research has found that Arabidopsis AtROP10 is a negative regulator of multiple physiological responses in ABA signaling, including stomatal closure, seed dormancy and germination, the inhibition of root growth, and gene expression [25,26].
The function of ROP is not only restricted to plant physiological process regulation but is also important for plant responses to pathogen invasion.The overexpression of constitutive-activated GhROP6 can increase the contents of jasmonic acid-isoleucine and lignin in transgenic arabidopsis to enhance resistance to Verticillium wilt [27].The activation of OsRac1 affects the expression of related defense genes to enhance rice resistance to rice blast and bacterial blight [15], and OsRac1 and the transcription activator RAI1 jointly participate in resistance to rice blast disease [28].There are also reports that OsRac4, OsRac5, and OsRac6 negatively regulate the PTI defense response to rice [29,30].Barley ROP protein (RACB) can block the invasion of Blumeria graminis into plant tissues [31].In addition, ROP can also regulate the establishment of the ETI resistance mechanism.The overexpression of DNOsRacl in tobacco can significantly inhibit the production of ROS and HR, which are induced by the resistance gene Pto [32].StRac1 positively regulates potato resistance to Phytophthora infestans by mediating the H 2 O 2 content [33].ROP-mediated H 2 O 2 production can be inhibited by DPI (NADPH oxidase inhibitor), indicating that NADPH oxidases, also called respiratory burst oxidase homologs (RBOHs), are located downstream of small G proteins and regulate the production of H 2 O 2 [34,35].In plant-pathogen interactions, AtRBOHD and AtRBOHF are associated with apoplastic ROS generation to regulate their resistance [36,37].CAOsRac1 can enhance pathogen-associated molecular pattern (PAMP)induced ROS accumulation and resistance to pathogens in rice, and OsRac1 can both induce the accumulation of ROS and inhibit the activity of ROS scavenging enzymes [15,38].Similarly, the overexpression of the activating CAHvRac1 in barley can resist infection of powdery mildew by accumulating ROS [39].
Hormone signal transduction is also one of the main pathways of small G protein resistance.The negative regulatory effect of ROPs on ABA response is concluded through the study of plant guard cells.In the absence of exogenous ABA, the overexpression of CA ROP6 can inhibit ABA-induced stomatal closure in wild-type arabidopsis plants.
However, the expression of DN-ROP6 can cause stomatal closure in the leaves of wild-type and ABA mutant abi-1 [40,41].ROP9 and ROP10 have also been shown to be negative regulators of the ABA response [42].Treatment with ABA leads to the inactivation of AtROP3, the AtROP10 promoter in Arabidopsis [40,43].PYLs, PP2Cs, and SnRK2s are the three core components of the ABA signaling pathway [44].ABI5 is an important leucine zipper (bZIP) transcription factor in the ABA-mediated signal transduction pathway [45].However, some studies have shown that small G proteins play a negative regulatory role in disease resistance.Zhang et al. (2014) found that heterologous overexpression of the inactivated DN-ROP1 of Arabidopsis in potatoes can significantly enhance their resistance to Phytophthora infestans, which is associated with the NADPH oxidase-mediated accumulation of H 2 O 2 [46].However, the molecular mechanism of gene resistance to C. cassiicola in the signaling pathway of cucumber CsROPs has not been explored.
In this study, two CsROPs were cloned from cucumber leaves.A sequence analysis of the CsROPs revealed that one structure was similar to that of Arabidopsis ROP5, named CsROP5 (Cucsa.322750),and the other structure was similar to that of Arabidopsis ROP10, named CsROP10 (Cucsa.197080).The transient CsROP5/CsROP10 overexpression reduced the host resistance response to C. cassiicola, whereas transient CsROP5/CsROP10 silencing enhanced the resistance of cucumber to C. cassiicola.Furthermore, changes in the transcript levels of pathogenesis-related proteins, ABA-signaling-pathway-associated genes, or ROS-associated genes mediated via CsROP10 silencing enhance cucumber resistance to C. cassiicola.CsROP5 silencing regulated the expression of ROS-signaling-related genes and pathogenesis-related proteins, enhancing the defense response to C. cassiicola.Therefore, we speculated that CsROP5 and CsROP10 may be negative modulators and indirectly involved in cucumber defense responses to C. cassiicola through multiple signaling networks.

Plant Materials
The cucumber cultivar C. sativus L. cv.Xintaimici was used in this experiment.Xintaimici is susceptible to C. cassiicola.All cucumber plants were grown in a nutrient compound of peatsoil/vermiculite (1/2, v/v) at 25 • C, in a 16 h/8 h light/dark cycle.

Pathogens and Inoculation
C. cassiicola of cucumber was streaked on PDA medium (10 g•L −1 D-glucose, 10 g•L −1 agar, and 200 g•L −1 potatoes).Solid clumps of C. cassiicola were inoculated on transgenic cucumber cotyledons in a greenhouse.The optimal cultivation temperature for C. cassiicola is 25 • C.

Sequence Analysis
The candidate cDNA sequences of CsROP5 (Cucsa.322750)and CsROP10 (Cucsa.197080)from susceptible cucumber leaves were cloned.The primers listed in Supplementary Table S2 were designed for the cDNA sequence of the DNAMAN.The CsROP5 and CsROP10 sequences were obtained from the BLAST (http://cucurbitgenomics.org/BLAST, accessed on 5 June 2022) results for similarity analysis (Figures S1 and S2).The conserved domains of CsROP5 and CsROP10 were compared with Arabidopsis thaliana ROP family members and human ROP family members (Figure 1).

Quantitative RT-qPCR
The expression patterns of candidate genes were analyzed with RT-qPCR using a Rocha instrument.The internal reference gene we selected was the cucumber actin gene [47].Supplementary Table S1 shows the specific primers.

Virus-Induced Gene Silencing (VIGS)
Using the pTRV (tobacco rattle virus)-based VIGS technique, we constructed a knockdown vector of the CsROP5 and CsROP10 genes.The two sequences were CsROP5 (from nucleotide 378 to 591 in the CsROP5 cDNA sequence) and CsROP10 (from nucleotide 429 to 639 in the CsROP10 cDNA sequence), which were subcloned in a sense orientation into the pTRV2 vector.The gene primers are shown in Table S2.
Agrobacterium tumefaciens strain EHA105 was placed in YEP medium with 100 mg•L −1 of rifampicin and 50 mg•L −1 of kanamycin and grown overnight at 28 °C.Then, 200 μL of the solution cultured overnight was absorbed and transferred to 20 mL of YEP medium with 100 mg•L −1 of rifampicin and 50 mg•L −1 of kanamycin.The above solution was incubated with shock until reaching OD600 = 0.8-1.0.

Quantitative RT-qPCR
The expression patterns of candidate genes were analyzed with RT-qPCR using a Rocha instrument.The internal reference gene we selected was the cucumber actin gene [47].Supplementary Table S1 shows the specific primers.

Virus-Induced Gene Silencing (VIGS)
Using the pTRV (tobacco rattle virus)-based VIGS technique, we constructed a knockdown vector of the CsROP5 and CsROP10 genes.The two sequences were CsROP5 (from nucleotide 378 to 591 in the CsROP5 cDNA sequence) and CsROP10 (from nucleotide 429 to 639 in the CsROP10 cDNA sequence), which were subcloned in a sense orientation into the pTRV2 vector.The gene primers are shown in Table S2.
Agrobacterium tumefaciens strain EHA105 was placed in YEP medium with 100 mg•L −1 of rifampicin and 50 mg•L −1 of kanamycin and grown overnight at 28 • C.Then, 200 µL of the solution cultured overnight was absorbed and transferred to 20 mL of YEP medium with 100 mg•L −1 of rifampicin and 50 mg•L −1 of kanamycin.The above solution was incubated with shock until reaching OD 600 = 0.8-1.0.

Construction of the Overexpressing Vector
The CsROP5 and CsROP10 cDNA sequences with the stop codon removed were amplified, and then PCR products were inserted between the SalI and BamHI cleavage sites of the pRI101-GFP vector.The expression of two target genes was induced in cucumber cotyledons under the control of the CaMV 35S promoter.The gene primers are shown in Table S2.Agrobacterium tumefaciens strain EHA105 was placed in YEP medium with shock until reaching OD 600 = 0.8-1.0.The detailed steps are the same as described above.

Histochemical Analysis
The H 2 O 2 and O 2 • − levels of the cucumber cotyledons were assessed using 3,3 ′ -diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) [49].The H 2 O 2 test was performed by soaking the cucumber cotyledon in 1 mg•mL −1 DAB for 8 h.The O 2 − test was performed by soaking the cucumber cotyledon in 0.1% NBT for 5 h.Then, ethanol/lactic acid/glycerol (3:1:1) was added to the cucumber cotyledons, and they were boiled for 20 min, transferred to 95% ethanol, and stored at 4 • C.

Cloning and Sequence Analysis of CsROP5 and CsROP10
Two full-length cDNA sequences of CsROP5 and CsROP10 were cloned from cucumber leaves.The analysis showed that the CsROP5 cDNA encoded a 21.56 kDa protein with 197 amino acid residues and the CsROP10 cDNA encoded a 23.42 kDa protein with 210 amino acid residues (Figures S1 and S2).An analysis of the protein sequence showed that CsROP5 and CsROP10 contain ROP-conserved domains (Figure 1).The CsROP5 and CsROP10 proteins were quite similar to the Arabidopsis thaliana ROP family members, which contained the highest conservation rate, including five sequence motifs (G1-G5) that were composed of a central six-stranded β-sheet (β1-β6) surrounded by five α-helices (α1-α5), the helix αi of the Rho insert, and a short helical structure (η1) upstream of the insert (Figure 1).Additionally, the sequences of CsROP5 and CsROP10 were clearly distinct from Rac1 as well as RhoA.The sequences of CsROP5 and CsROP10 were significantly distinct from the Rac1 and RhoA sequences in that there was a possible recognition site (SYR) for serine/threonine kinases, which was produced by the conserved arginine present in the downstream ROP protein of switch II [19].

CsROP5 and CsROP10 Silencing in Cucumber Enhances Its Resistance to C. cassiicola
In order to investigate whether the transcript levels of CsROP5 and CsROP10 affect the resistance of cucumber to C. cassiicola, the VIGS technique was used to knockdown the CsROP5/CsROP10 transcript in the susceptible cultivar Xintaimici (Figure 2).The 3 ′ -terminal fragment specific to CsROP5 and CsROP10 was inserted into the restriction site of pTRV2, which generated the recombinant structures of pTRV2-CsROP5 and pTRV2-CsROP10 (Figure 2A).After 7 days of inoculation with pTRV2-CsROP5, pTRV2-CsROP10, and TRV:00 (as a control), the cucumber cotyledons of the infected plants showed chlorotic mosaic symptoms (Figure 2B), proving that these inoculated plants were successfully infected with TRV.Meanwhile, the RT-qPCR confirmed the effective silencing of CsROP5 and CsROP10 (Figure 2C).These results indicate that CsROP5 and CsROP10 were almost completely unexpressed in cucumber plants after silencing.Subsequently, C. cassiicola was inoculated on cucumber cotyledons to evaluate the defense resistance of CsROP5 and CsROP10 (Figure 3).To investigate the role of CsROP5 and CsROP10 in the defense against C. cassiicola, we tested the transgenic cucumber cotyledons after 5 days of pathogen infection.The results of the lesion size measurement showed that the cucumber cotyledons were comparable between the non-injected cucumber cotyledons and the cucumber cotyledons inoculated with TRV:00.However, the lesion areas on the cotyledons of CsROP5-silencing and CsROP10-silencing plants were 0.40-0.45cm, while the average lesion areas of the non-injected cucumber cotyledons and the TRV:00-injected groups were 0.60-0.63cm.These results show that CsROP5 and CsROP10 silencing in Xintaimici plants enhanced their defense response to C. cassiicola.Subsequently, C. cassiicola was inoculated on cucumber cotyledons to evaluate the defense resistance of CsROP5 and CsROP10 (Figure 3).To investigate the role of CsROP5 and CsROP10 in the defense against C. cassiicola, we tested the transgenic cucumber cotyledons after 5 days of pathogen infection.The results of the lesion size measurement showed that the cucumber cotyledons were comparable between the non-injected cucumber cotyledons and the cucumber cotyledons inoculated with TRV:00.However, the lesion areas on the cotyledons of CsROP5-silencing and CsROP10-silencing plants were 0.40-0.45cm, while the average lesion areas of the non-injected cucumber cotyledons and the TRV:00-injected groups were 0.60-0.63cm.These results show that CsROP5 and CsROP10 silencing in Xintaimici plants enhanced their defense response to C. cassiicola.Subsequently, C. cassiicola was inoculated on cucumber cotyledons to evaluate the defense resistance of CsROP5 and CsROP10 (Figure 3).To investigate the role of CsROP5 and CsROP10 in the defense against C. cassiicola, we tested the transgenic cucumber cotyledons after 5 days of pathogen infection.The results of the lesion size measurement showed that the cucumber cotyledons were comparable between the non-injected cucumber cotyledons and the cucumber cotyledons inoculated with TRV:00.However, the lesion areas on the cotyledons of CsROP5-silencing and CsROP10-silencing plants were 0.40-0.45cm, while the average lesion areas of the non-injected cucumber cotyledons and the TRV:00-injected groups were 0.60-0.63cm.These results show that CsROP5 and CsROP10 silencing in Xintaimici plants enhanced their defense response to C. cassiicola.

CsROP5 and CsROP10 Overexpression in Cucumber Cotyledons Impairs Resistance to C. cassiicola
For further evaluation of the roles of CsROP5 and CsROP10 in the defense responses, two overexpression vectors containing pRI101:GFP-CsROP5 and pRI101:GFP-CsROP10 were generated.The constructed recombinant plasmids (GFP::CsROP5/GFP::CsROP10) and empty plasmids (GFP::00) were transformed into Agrobacterium tumefaciens to be transferred into cucumber cotyledons (Figure 4).The PCR analysis using chimeric primers between the CsROP5/CsROP10 and GFP fusion genes showed that the introduced transgene could be detected in the cucumber cotyledons (Figure 4A; Figure 4B).Meanwhile, the RT-qPCR results show that the transcript abundances of CsROP5/CsROP10 were significantly increased compared to the GFP::00-injected plants (Figure 4C).Our results demonstrate that CsROP5 and CsROP10 were successfully transiently overexpressed in cucumber cotyledons.
Biology 2024, 13, 308 7 of 16 For further evaluation of the roles of CsROP5 and CsROP10 in the defense responses, two overexpression vectors containing pRI101:GFP-CsROP5 and pRI101:GFP-CsROP10 were generated.The constructed recombinant plasmids (GFP::CsROP5/GFP::CsROP10) and empty plasmids (GFP::00) were transformed into Agrobacterium tumefaciens to be transferred into cucumber cotyledons (Figure 4).The PCR analysis using chimeric primers between the CsROP5/CsROP10 and GFP fusion genes showed that the introduced transgene could be detected in the cucumber cotyledons (Figure 4A; Figure 4B).Meanwhile, the RT-qPCR results show that the transcript abundances of CsROP5/CsROP10 were significantly increased compared to the GFP::00-injected plants (Figure 4C).Our results demonstrate that CsROP5 and CsROP10 were successfully transiently overexpressed in cucumber cotyledons.Following inoculation with C. cassiicola for 5 d, comparing the non-injected and GFP::00-injected plants, the defense resistance levels to C. cassiicola of CsROP5/CsROP10overexpressing cucumber cotyledons had significantly reduced (Figure 5).The lesion size measurements in the cotyledons of CsROP5-overexpressing and CsROP10-overexpressing plants infected with C. cassiicola ranged from 0.79 to 0.81 cm, while the average lesion areas of the non-injected cucumber cotyledons and the GFP::00-injected groups were 0.60-0.62cm.The above results show that transient overexpression of CsROP5 and CsROP10 reduced cucumber resistance to C. cassiicola.These results indicate that CsROP5 and CsROP10 negatively regulate cucumber resistance response to C. cassiicola.Following inoculation with C. cassiicola for 5 d, comparing the non-injected and GFP::00-injected plants, the defense resistance levels to C. cassiicola of CsROP5/CsROP10overexpressing cucumber cotyledons had significantly reduced (Figure 5).The lesion size measurements in the cotyledons of CsROP5-overexpressing and CsROP10-overexpressing plants infected with C. cassiicola ranged from 0.79 to 0.81 cm, while the average lesion areas of the non-injected cucumber cotyledons and the GFP::00-injected groups were 0.60-0.62cm.The above results show that transient overexpression of CsROP5 and CsROP10 reduced cucumber resistance to C. cassiicola.These results indicate that CsROP5 and CsROP10 negatively regulate cucumber resistance response to C. cassiicola.

CsROP5 and CsROP10 Modulate the Expression Levels of Defense-Related Genes after C. cassiicola Challenge in Transgenic Plants
To investigate whether CsROP5/CsROP10 expression influenced defense-related genes in cucumber response to C. cassiicola, the expression patterns of two pathogenesisrelated genes were analyzed in transgenic cucumbers (Figure 6).The results show that the expression of CsPR2 was significantly increased in CsROP5/CsROP10-silencing cotyledons compared to TRV::00-infected plants, and the transcript level of CsPR2 was only decreased in CsROP5-overexpressing cucumber cotyledons after C. cassiicola inoculation for 5 days.Additionally, the transcript levels of CsPR3 were also significantly induced in CsROP5/CsROP10-silencing cotyledons, and the transcript levels of CsPR3 were significantly declined in CsROP5/CsROP10-overexpressing cucumber cotyledons.In summary, we speculate that CsRP might participate in the defense reaction through the CsROP5/CsROP10-induced defense pathway.

ROS Homoeostasis Is Crucial for CsROP5-and CsROP10-Mediated Resistance against C. cassiicola
To investigate whether CsROP5 and CsROP10 mediate the homeostasis between ROS scavenging and production to affect cucumber resistance to C. cassiicola, we conducted the following experiments (Figure 7).H2O2 and O2 − were detected using the DAB and NBT

CsROP5 and CsROP10 Modulate the Expression Levels of Defense-Related Genes after C. cassiicola Challenge in Transgenic Plants
To investigate whether CsROP5/CsROP10 expression influenced defense-related genes in cucumber response to C. cassiicola, the expression patterns of two pathogenesis-related genes were analyzed in transgenic cucumbers (Figure 6).The results show that the expression of CsPR2 was significantly increased in CsROP5/CsROP10-silencing cotyledons compared to TRV::00-infected plants, and the transcript level of CsPR2 was only decreased in CsROP5-overexpressing cucumber cotyledons after C. cassiicola inoculation for 5 days.Additionally, the transcript levels of CsPR3 were also significantly induced in CsROP5/CsROP10silencing cotyledons, and the transcript levels of CsPR3 were significantly declined in CsROP5/CsROP10-overexpressing cucumber cotyledons.In summary, we speculate that CsRP might participate in the defense reaction through the CsROP5/CsROP10-induced defense pathway.

CsROP5 and CsROP10 Modulate the Expression Levels of Defense-Related Genes after C. cassiicola Challenge in Transgenic Plants
To investigate whether CsROP5/CsROP10 expression influenced defense-related genes in cucumber response to C. cassiicola, the expression patterns of two pathogenesisrelated genes were analyzed in transgenic cucumbers (Figure 6).The results show that the expression of CsPR2 was significantly increased in CsROP5/CsROP10-silencing cotyledons compared to TRV::00-infected plants, and the transcript level of CsPR2 was only decreased in CsROP5-overexpressing cucumber cotyledons after C. cassiicola inoculation for 5 days.Additionally, the transcript levels of CsPR3 were also significantly induced in CsROP5/CsROP10-silencing cotyledons, and the transcript levels of CsPR3 were significantly declined in CsROP5/CsROP10-overexpressing cucumber cotyledons.In summary, we speculate that CsRP might participate in the defense reaction through the CsROP5/CsROP10-induced defense pathway.

ROS Homoeostasis Is Crucial for CsROP5-and CsROP10-Mediated Resistance against C. cassiicola
To investigate whether CsROP5 and CsROP10 mediate the homeostasis between ROS scavenging and production to affect cucumber resistance to C. cassiicola, we conducted the following experiments (Figure 7).H2O2 and O2 − were detected using the DAB and NBT

ROS Homoeostasis Is Crucial for CsROP5-and CsROP10-Mediated Resistance against C. cassiicola
To investigate whether CsROP5 and CsROP10 mediate the homeostasis between ROS scavenging and production to affect cucumber resistance to C. cassiicola, we conducted the following experiments (Figure 7).− was lower in the CsROP5/CsROP10-overexpressing cucumber than that in the GFP::00 cucumber.Overall, the accumulation of H 2 O 2 and O 2 − was significantly higher in the silencing cucumber than that in the overexpressing cucumber after C. cassiicola infection.Following C. cassiicola inoculation for 5 days, two ROS-formation-related genes, CsRbohD (Cucsa.340760)and CsRbohF (Cucsa.107010),were closely linked to the transcript levels of CsROP5 and CsROP10 in the cucumber cotyledons (Figure 7A).The transcript levels of the above two genes were significantly higher in the CsROP10-silencing plants than in the TRV::00 plants and were lower in the CsROP10overexpressing plants.However, the transcript levels of CsRbohD and CsRbohF were only elevated in the CsROP5-silencing plants compared to the controls, but nothing changed in the CsROP5-overexpressing plants.These results imply that ROS signaling was triggered in the CsROP10-silencing plants, mediating their resistance to C. cassiicola.staining methods in transgenic plants (Figure 7B).After 12, 24, and 48 h of inoculation with C. cassiicola, the accumulation of H2O2 and O2 − in CsROP5/CsROP10-silencing plants was increased compared to that in TRV::00 plants, with more brown and blue spots on the leaves.However, little H2O2 and O2 − was detected at 12 h with C. cassiicola inoculation in the plants and the GFP::00 plants.After 24 and 48 h, the accumulation of H2O2 and O2 − was lower in the CsROP5/CsROP10-overexpressing cucumber than that in the GFP::00 cucumber.Overall, the accumulation of H2O2 and O2 − was significantly higher in the silencing cucumber than that in the overexpressing cucumber after C. cassiicola infection.Following C. cassiicola inoculation for 5 days, two ROS-formation-related genes, CsRbohD (Cucsa.340760)and CsRbohF (Cucsa.107010),were closely linked to the transcript levels of CsROP5 and CsROP10 in the cucumber cotyledons (Figure 7A).The transcript levels of the above two genes were significantly higher in the CsROP10silencing plants than in the TRV::00 plants and were lower in the CsROP10-overexpressing plants.However, the transcript levels of CsRbohD and CsRbohF were only elevated in the CsROP5-silencing plants compared to the controls, but nothing changed in the CsROP5overexpressing plants.These results imply that ROS signaling was triggered in the CsROP10-silencing plants, mediating their resistance to C. cassiicola.

CsROP5 and CsROP10 Modulate Abscisic Acid (ABA)-Signaling Components
ROP GTPases are important signaling proteins that regulate ABA-related responses [21,40,43].Therefore, the transcript levels of genes related to the ABA signaling pathway were detected in CsROP5/CsROP10-silencing plants and CsROP5/CsROP10overexpressing plants after C. cassiicola inoculation for 5 days (Figure 8).The RT-qPCR analysis showed that the transcript levels of CsPYL2 (JF789829), CsSnRK2.2 (JN566071), and CsABI5 (XM_004149176.2) were increased in the CsROP10-silencing plants but decreased in the CsROP10-overexpressing plants.However, the transcript level of CsPP2C (JN566067) showed the opposite trend, resulting in a significantly suppressed transcript level of CsPP2C in the CsROP10-silencing plants and a more markedly induced level in the CsROP10-overexpressing plants than in the control plants.In addition, we also conducted transcript-level detection of genes related to the ABA pathway in CsROP5 transient transgenic plants.Compared to the control levels, the transcript level of CsPP2C was decreased in the CsROP5-silencing plants and increased in the CsROP5-overexpressing plants.The transcript level of CsPYL2 was only improved in the CsROP5-overexpressing plants.However, there was almost no change in the transcript levels of CsABI5 and CsS-nRK2.2 in the CsROP5 transient transgenic plants.These results suggest that CsROP10 negatively regulated the transcript levels of CsPYL2, CsSnRK2.2, and CsABI5 and positively regulated the transcript level of CsPP2C.Together, these results suggest that CsROP10 was a negative regulator and improved the transcript level of ABA-signaling component genes to enhance the cucumber defense response to C. cassiicola.However, the expression regulation of ABA signaling pathway genes was not directly related to the transcript level of CsROP5, indicating that there might be other defense mechanisms of CsROP5 in the cucumber-C.cassiicola interaction.
in the CsROP10-overexpressing plants.However, the transcript level of CsPP2C (JN566067) showed the opposite trend, resulting in a significantly suppressed transcript level of CsPP2C in the CsROP10-silencing plants and a more markedly induced level in the CsROP10-overexpressing plants than in the control plants.In addition, we also conducted transcript-level detection of genes related to the ABA pathway in CsROP5 transient transgenic plants.Compared to the control levels, the transcript level of CsPP2C was decreased in the CsROP5-silencing plants and increased in the CsROP5-overexpressing plants.The transcript level of CsPYL2 was only improved in the CsROP5-overexpressing plants.However, there was almost no change in the transcript levels of CsABI5 and CsSnRK2.2 in the CsROP5 transient transgenic plants.These results suggest that CsROP10 negatively regulated the transcript levels of CsPYL2, CsSnRK2.2, and CsABI5 and positively regulated the transcript level of CsPP2C.Together, these results suggest that CsROP10 was a negative regulator and improved the transcript level of ABA-signaling component genes to enhance the cucumber defense response to C. cassiicola.However, the expression regulation of ABA signaling pathway genes was not directly related to the transcript level of CsROP5, indicating that there might be other defense mechanisms of CsROP5 in the cucumber-C.cassiicola interaction.

Discussion
The CDS regions of two CsROP genes, namely CsROP5 and CsROP10, were identified and cloned in cucumber cotyledons.A sequence analysis showed that CsROP5 and

Discussion
The CDS regions of two CsROP genes, namely CsROP5 and CsROP10, were identified and cloned in cucumber cotyledons.A sequence analysis showed that CsROP5 and CsROP10 were distinct from other RHO GTPases in several aspects.First, the highly conserved effector domain (domain II) of CsROP5 and CsROP10 contained the highly conserved effector domain (domain I and domain II).Second, there were RHO insertion regions (structural domains V) in CsROP5 and CsROP10, which interacted with the RHO effectors [50].In addition, the sequence analysis showed that CsROP5 and CsROP10 contained two putative serine/threonine phosphorylation sites, SYR and SSK.The above domain modules were consistent with the ROPs of Arabidopsis [17].It has been confirmed that Arabidopsis, rice, maize, and other species contain multiple ROP proteins [20,51,52].
The ROP family also has diversity, with the highly variable region (HVR) defined by the region with the most variable C-terminus of the ROP protein [53][54][55].In our study, an HVR similar to that of ROP family genes was predicted in cucumber plants.Meanwhile, a functional analysis found that barley Rac/ROP G-protein family members were susceptible to the powdery mildew fungus [55].The study demonstrated that CsROP5 and CsROP10 genes might mediate pathogen in cucumber.
Increasing evidence shows that ROPs are an indispensable regulatory factor in plant immune signaling [56].OsRac4 and OsRac5 are negative regulators of blast resistance [30].Three members of the Rac/ROP family in barley-HvRACB, HvRAC3, and HvROP6-play a negative regulatory role in powdery mildew fungus [55,57].Currently, the role of the ROP gene is unclear in cucumber-C.cassiicola interactions.Due to constriction in cucumber transgenic technology, research on the molecular mechanism of ROP gene resistance to disease has been hindered in cucumbers.At present, the experimental method of transient agrobacterium infection of cucumber cotyledons has matured [48].This study on CsROP5 silencing and CsROP10 overexpression was further carried out using an experimental method of agrobacterium infiltration into cucumber cotyledons.The infection assays insinuated that CsROP5 and CsROP10 played an important role in the resistance of cucumber to C. cassiicola.CsROP5/CsROP10-silencing plants exhibited strongly enhanced resistance to C. cassiicola, while CsROP5/CsROP10-overexpressing plants showed significantly reduced resistance to C. cassiicola.In addition, ROS accumulation is required for the plant defense mechanism when part of a plant is subjected to C. cassiicola attack [58].In our research, ROP might have triggered the outbreak of ROS, thereby enhancing the resistance to C. cassiicola infection in cucumber cotyledons.ROS accumulation also occurred earlier in the CsROP5/CsROP10silencing plants than in the CsROP5/CsROP10-overexpressing plants after C. cassiicola infection.Thus, we preliminarily speculate that the responses of the plants to C. cassiicola stress may have revealed CsROP5 and CsROP10 as negative regulatory factors.
As is well known, the most influential enzyme for ROS production is NADPH oxidase.The interaction between OsRac1 and the N-terminal domain of OsrbohB promotes the accumulation of ROS in rice [59,60].The interaction between DN-AtROP1 and StrbohD (a potato NADPH oxidase) induces H 2 O 2 accumulation to negatively regulate resistance to potato Phytophthora infestans [46].In similar cases, ROS-producing NADPH oxidases are affected by AtrbohD and AtrbohF genes in response to pathogen infection [36,37,61].The overexpression of CA OsRac1 can correspondingly increase the level of ROS and cause a response similar to HR, thereby increasing the resistance of rice to Magnaporthe oryzae and Blumeria graminis [39].Furthermore, we found that the expressions of CsRbohD and CsRbohF were increased in CsROP5/CsROP10-silencing cucumbers, whereas the expressions of CsRbohD and CsRbohF were suppressed in CsROP10-overexpressing cucumbers.Compared to the control, the accumulation of H 2 O 2 and O 2 − was reduced in CsROP10-silencing cucumbers after C. cassiicola inoculation, and similar results were observed in CsROP5silencing cucumbers.Therefore, it is highly likely that small G proteins have similar ways of generating ROS in cucumbers, and the differences in the binding affinity or activation ability of CsROP5/CsROP10 to the cucumber RBOHB homologs led to differences in ROS production.Additionally, these results suggest that ROS signaling might be pre-activated in CsROP5/CsROP10-silencing cucumbers to enhance defense resistance against C. cassiicola.
In addition, pathogenesis-related proteins (PRs), including β-1,3-glucanase (PR2) and chitinase (PR3) [62,63], are a type of protein produced in plants under pathogen infection to regulate defense responses.Previous research has demonstrated that the expression of arabidopsis pathogenesis-related proteins improve resistance to B. cinerea interactions [64].The Rac/ROP plays a key role in defense systems, such as regulating the expression of pathogen-related genes and lignin biosynthesis [65][66][67][68].In our study, the expression patterns of defense-related CsPR2 and CsPR3 were altered in CsROP5/CsROP10induced disease resistance.The treatment of CsROP5/CsROP10-silencing plants primed defense responses against C. cassiicola in cucumber plants by inducing higher expression of the defense-related genes CsPR2 and CsPR3.This finding indicates that other resistance mechanisms exist in response to C. cassiicola; CsROP5/CsROP10 primarily mediated the defense resistance via the PR proteins.In summary, CsROP5-silencing and CsROP10silencing regulated ROS signaling pathways may jointly activate downstream pathogenesisrelated gene expression against C. cassiicola.
ABA is a key hormone that regulates plant responses to many types of adaptive stress [69].ABA can actively regulate stomatal closure against pathogens or induce callose deposition when pathogens evade the first line of defense, thereby regulating plant defense in the early stages of infection [70,71].In addition, research on plant guard cells has shown that ROPs can negatively regulate ABA signaling [40].Research has found that ROP9 and ROP10 are negative regulators of ABA reactions.Treatment with ABA resulted in the inactivation of AtROP3 (AtRac1) and AtROP10 promoters in Arabidopsis [40,43].It was found that CsPYL2, CsPP2C2, and CsSnKR2.2 are involved in transducing the ABA signal in regulating plant development and drought stress [44].A comprehensive expression analysis of ABA signal transduction genes in cucumber revealed that CsROP10 inhibited the expressions of CsPYL2 and CsSnKR2.2 and facilitated the expression of CsPP2C2 in response to C. cassiicola.These results are consistent with the results of Ulferts, who reported that ABA negatively interfered with the basal defense of barley against Magnaporthe oryzae [72].In addition, compared to the control, the transcript level of CsABI5 was upregulated by seven-fold at 5 dpt under C. cassiicola in the CsROP10-silencing plant, although the transcript level of CsABI5 was significantly downregulated in the CsROP10-silencing plant (Figure 8).Similar to these findings, CsABI5 genes were highly induced in CsMLO1-silencing cucumber cotyledons after C. cassiicola infestation [9].The above results also coincide with the interaction effect in CsROP10 and C. cassiicola via mediating the ABA signaling pathway.ABA binds to receptors (RCAR/PPYR/PYL), leading to phosphorylating PP2C and activating protein kinase.Additionally, more and more evidence suggests that ROP and ABA form a negative feedback loop, where ABA signaling inhibits ROP activation and ROP signaling inhibits ABA response; for example, ROP10 and ROP11 inhibit ABA signal transduction through physical interactions with ABA negative regulatory factors, ABA insensitivity 1 (ABI1), and ABI2 PP2C phosphatase [73,74].Therefore, we speculate that CsROP10 also may interact with ABI proteins to inhibit ABA signaling pathways in cucumber plants.The elevated kinase activates NADPH oxidase and elevates ROS production [75].Furthermore, RBOHC is activated by activated ROPs, and this change leads to the generation of ROS, which causes changes in the intracellular and extracellular pH and intracellular Ca 2+ levels [76,77].Research has found that ROS-and ABA-signaling-related genes are related to plant immunity, thereby improving the resistance of cucumber to C. cassiicola in CsMLO1-silencing plants [9].Thus, our results suggest that CsROP10 participates in pathogen resistance by employing the ABA-mediated ROS signaling pathway and the R gene pathway associated with CsROP10.However, the change in the transcript level of CsROP5 had no regular effect on the expression of ABA-related genes.This finding further suggests that the involvement of CsROP5 in the defense of C. cassiicola may be related to ROS signaling and defense-related genes.

Conclusions
In conclusion, two cucumber genes, CsROP5 and CsROP10, negatively contribute to the defense response to C. cassiicola.CsROP10 can regulate the expression of ABAsignaling-related genes, thereby promoting the production and accumulation of ROS genes, subsequently modulating the expression of pathogenesis-related proteins and ultimately leading to enhanced resistance to C. cassiicola.Cucumber CsROP5 regulated the expression of ROS-signaling-related genes and pathogenesis-related proteins, enhancing the defense response to C. cassiicola.Therefore, this study indicates that the plant defense response is mediated by the interaction of the CsROP5 and CsROP10 genes between cucumber and C. cassiicola.

Figure 3 .
Figure 3. Demonstrated enhanced resistance to C. cassiicola in silenced plants.(A) The phenotypes of cucumber cotyledons 5 days after inoculation with C. cassiicola.(B) The lesion size was determined at cucumber cotyledons.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 3 .
Figure 3. Demonstrated enhanced resistance to C. cassiicola in silenced plants.(A) The phenotypes of cucumber cotyledons 5 days after inoculation with C. cassiicola.(B) The lesion size was determined at cucumber cotyledons.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 3 .
Figure 3. Demonstrated enhanced resistance to C. cassiicola in silenced plants.(A) The phenotypes of cucumber cotyledons 5 days after inoculation with C. cassiicola.(B) The lesion size was determined at cucumber cotyledons.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 5 .
Figure 5. Identification of disease resistance of CsROP5-overexpressing and CsROP10-overexpressing plants.(A) The phenotypes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.(B) The lesion sizes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 6 .
Figure 6.Relative mRNA transcript levels of defense-related genes of CsROP5 and CsROP10 transgenic plants as determined by RT-qPCR.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 5 .
Figure 5. Identification of disease resistance of CsROP5-overexpressing and CsROP10-overexpressing plants.(A) The phenotypes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.(B) The lesion sizes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Biology 2024, 13 , 308 8 of 16 Figure 5 .
Figure 5. Identification of disease resistance of CsROP5-overexpressing and CsROP10-overexpressing plants.(A) The phenotypes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.(B) The lesion sizes of cucumber cotyledons at 5 days after inoculation with C. cassiicola.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 6 .
Figure 6.Relative mRNA transcript levels of defense-related genes of CsROP5 and CsROP10 transgenic plants as determined by RT-qPCR.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).

Figure 6 .
Figure 6.Relative mRNA transcript levels of defense-related genes of CsROP5 and CsROP10 transgenic plants as determined by RT-qPCR.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).
H 2 O 2 and O 2 − were detected using the DAB and NBT staining methods in transgenic plants (Figure 7B).After 12, 24, and 48 h of inoculation with C. cassiicola, the accumulation of H 2 O 2 and O 2 − in CsROP5/CsROP10-silencing plants was increased compared to that in TRV::00 plants, with more brown and blue spots on the leaves.However, little H 2 O 2 and O 2 − was detected at 12 h with C. cassiicola inoculation in the CsROP5/CsROP10-overexpressing plants and the GFP::00 plants.After 24 and 48 h, the accumulation of H 2 O 2 and O 2

Figure 7 .
Figure 7. ROS signaling was modulated in transgenic plants after inoculation with C. cassiicola.(A) Relative mRNA transcript levels of CsRbohD and CsRbohF in CsROP5/CsROP10 transgenic plants as determined by RT-qPCR.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).(B) DAB and NBT staining of H2O2 and O2 − generation in transgenic plants after CsROP5 and CsROP10 Agrobacterium infiltration.Data are means of three biological replicates of per variety.

Figure 7 .
Figure 7. ROS signaling was modulated in transgenic plants after inoculation with C. cassiicola.(A) Relative mRNA transcript levels of CsRbohD and CsRbohF in CsROP5/CsROP10 transgenic plants as determined by RT-qPCR.Asterisks indicate significant difference (Student's t-test, ** p < 0.01).(B) DAB and NBT staining of H 2 O 2 and O 2 − generation in transgenic plants after CsROP5 and CsROP10 Agrobacterium infiltration.

Figure 8 .
Figure 8. Relative mRNA transcript levels of ABA-related genes in CsROP5 and CsROP10 transgenic plants as determined by RT-qPCR.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, * p < 0.05 or ** p < 0.01).

Figure 8 .
Figure 8. Relative mRNA transcript levels of ABA-related genes in CsROP5 and CsROP10 transgenic plants as determined by RT-qPCR.Error bars represent standard deviations from three independent replicates.Asterisks indicate significant difference (Student's t-test, * p < 0.05 or ** p < 0.01).